We have used diverse fluorescence imaging approaches combined with quantitative analysis to investigate the characteristics of endomembrane organization in eukaryotic cells, including nonpolarized and polarized cell monolayers in tissue culture and in living embryos. Among the areas being investigated are: mitochondrial morphology and its regulation of cell cycle progression;the origin and dynamics of organelles;and cytoskeletal and endomembrane crosstalk in polarized epithelial cells and developing Drosophila syncitial blastoderm embryos. Related to our work characterizing mitochondria, we carried out live cell imaging experiments in cells stably expressing RFP targeted to the mitochondrial matrix to determine if there are changes in mitochondrial dynamism and function at different stages of the cell cycle. We found that mitochondria exhibit distinct morphological and physiological states at different stages of the cell cycle. In mitosis, mitochondria fragmented into hundreds of small units for partitioning into daughter cells at cytokinesis. Strikingly, at G1/S, mitochondria fused together into a single huge, dynamic filamentous system, unlike at any other cell cycle stage. Photobleaching of an area across this filamentous system revealed the mitochondrial matrix was continuous. The mitochondrial network also was electrically coupled and had a higher membrane potential than mitochondria at all other stages of the cell cycle. When the filamentous network or its membrane potential was disrupted, or its dynamics perturbed, cell cycle progression from G1 into S was arrested in a p53-dependent manner. Moreover, p21-overexpression, which induces a G1/S arrest, resulted in filamentous mitochondria with reduced matrix continuity and loss of electrical coupling. The data thus revealed that mitochondria dynamism and morphology undergo critical changes during the cell cycle that are sensed by the cell at G1/S to control cell cycle progression. We explored the origin and dynamics of organelles. One of these is the autophagosome. Autophagosomes form during autophagy, a highly conserved, bulk degradation pathway that is also involved in turnover of large aggregates and organelles within cells. In the initial step of this pathway, an isolation membrane forms in the cytoplasm through the activation of specific autophagy effectors. The membrane wraps around the protein aggregate or organelle to form a double membrane-bounded structure called the autophagosome. The autophagosome then targets to and fuses with the lysosome where the sequestered materials are degraded by various hydrolytic enzymes and recycled as amino acids for macromolecule synthesis and energy production. While emerging results have revealed the importance of autophagy in various biological and pathological processes, such as cellular remodeling, tumorogenesis and neurodegeneration, how this pathway operates is far from clear. We utilized various live cell imaging and molecular genetic approaches to investigate the membrane origin of autophagosomes and the signals that recruit substrates to this organelle. Our data revealed that the outer membrane of mitochondria serves as the membrane source during starvation-induced autophagy formation and maturation. Furthermore, ubiquitin modification acts as a targeting signal for delivery of small cytosolic proteins as well as larger organelles to autophagosomes. Epithelial cell migration requires coordination of two actin modules at the leading edge: one in the lamellipodium and one in the lamella. We used live-cell imaging and photoactivation approaches to determine how the two modules connect mechanistically to regulate directed edge motion. We discovered that the actin network of the lamellipodium evolves spatio-temporally into the lamella. This occurs during the retraction phase of edge motion, when myosin II redistributes to the lamellipodial actin and condenses it into an actin arc parallel to the edge. The new actin arc moves rearward, slowing down at focal adhesions in the lamella. Based on these results, we proposed a model for net edge extension in which nascent focal adhesions advance the site at which new actin arcs slow down and form the base of the next protrusion event. In this scheme, the actin arc serves as a structural element underlying the temporal and spatial connection between the lamellipodium and the lamella during directed cell motion. The final stage of cytokinesis is abscission, the cutting of the narrow membrane bridge connecting two daughter cells, has recently been shown to involve the endosomal sorting complex required for transport (ESCRT) machinery. To clarify how this machinery can drive abscission, we used structured illumination microscopy and time-lapse imaging to dissect the behavior of ESCRTs during abscission. Our data revealed that the ESCRT-I subunit tumor-susceptibility gene 101 (TSG101) and the ESCRT-III subunit charged multivesicular body protein 4b (CHMP4B) are sequentially recruited to the center of the intercellular bridge, forming a series of cortical rings. Late in cytokinesis, however, CHMP4B is acutely recruited to the narrow constriction site where abscission occurs. The ESCRT disassembly factor vacuolar protein sorting 4 (VPS4) follows CHMP4B to this site, and cell separation occurs immediately. That arrival of ESCRT-III and VPS4 correlates both spatially and temporally with the abscission event therefore suggests a direct role for these proteins in cytokinetic membrane abscission.